The production of proteins for industry or pharmacy is most commonly achieved by recombinant gene expression using heterologous host systems. The methylotrophic yeast Pichia pastoris is an important host system for heterologous gene expression. P. pastoris provides fast growth on simple media and is capable of providing most eukaryotic post translational modifications. Along with high capacities for protein production, P. pastoris is the only microbial expression host that provides fully humanized glycosylation (including sialylation) in engineered strains. Furthermore P. pastoris reaches exceptionally high cell densities (up to 130 g/l cell dry weight) and has high secretory capacities. As P. pastoris secretes only negligible amounts of endogenous protein, heterologous secreted proteins consist the vast majority of protein in the supernatant, thereby drastically facilitating purification and downstream processing.
When expressing a single protein, several factors influence the expression efficiency and thereby the yields. An important key factor to regulate expression is the promoter. The transcription strength of the promoter strongly influences the achieved yields. Strong promoters tend to give higher yields, but the effect is protein dependent. If other factors such as protein folding or post translational modifications are the expression limiting factor, too strong promoters might overburden the cellular machinery. In this case a weaker promoter might lead to better yields. In addition also the regulation of the promoter can influence the yields. Constitutive promoters provide more or less constant expression during the entire production process. However, the constitutive production of a protein of interest (POI) might interfere with the cellular metabolism and hamper growth, especially if the POI is toxic or difficult to express.
The coexpression of two (or more) proteins even further complicates expression efforts.
Dual gene coexpression is required when producing dimeric proteins (such as antibodies, consisting of a heavy and a light chain), an enzyme and a redox partner (such as Cytochrome P450 enzymes (CYP), which require a reductase (CPR) that delivers electrons) or when expressing a gene of interest (GOI) together with a folding helper (chaperone). Even more proteins have to be coexpressed for multimeric proteins and metabolic pathways.
The coexpressed proteins have to be expressed at the most suitable expression level, ratio and most favorable regulatory profile (constitutive, inducible or tunable expression simultaneously or in consecutive manner).
As for a single gene, the expression levels of multiple genes depend on their properties and might require strong or weak promoters to drive transcription. In addition, also the ratio of the coexpressed proteins is important. Depending on their natural role, they provide strongest activity/yields when expressed at equal levels, or one of the two proteins is required in several fold excess. For human P450s and the associated reductase it has been shown that a specific ratio of CYP to CPR is required for highest activity [1].
Furthermore, for the coexpression of two genes also the time frame has to be considered as the two proteins can either be expressed simultaneously or separately with one protein preceding expression. Especially in the coexpression of a helper protein, different time windows can be beneficial. Placing the helper protein under the control of a constitutive promoter and the GOI under a regulated/inducible promoter or consecutive induction provides the helper protein as a folding catalyst before starting GOI expression with an inductor. Using this approach, the helper protein expression precedes the GOI and is abundantly available when the GOI is expressed and can right away assist in folding.
Most gene coexpression efforts in P. pastoris have relied on the use of two separate vectors, with each vector providing one of the two genes [1,2].
The two vectors can either be cotransformed in the same strain [1] or transformed in two separate strains, which are subsequently mated, resulting in a strain carrying both genes [2].
Using two vectors also requires the use of two resistance markers. Concerning transfer of the vector, mating is relatively time consuming and requires at first the generation of single strains that express the GOIs. Cotransformation of the two vectors in one strain is linked with lower transformation efficiencies and requires immediate double selection on two antibiotics which can be detrimental in a case a critical protein is expressed constitutively.
Additionally, the two GOIs have also been placed on the same vector. In this case the same monodirectional promoter was cloned in front of the two GOIs [3]. This approach solves the problem of multiple resistance markers, but poses a problem as the same promoter sequence is present on the vector twice, which can lead to undesired recombination events. In contrast to open reading frames where the same amino acid sequence can be encoded by different gene sequences due to different codons there is no general concept to diversify the DNA sequence of promoters. Therefore mostly identical or completely different promoters with different properties are used to generate expression cassettes by individual fusions of coding regions with individual promoter sequences.
Concerning monodirectional promoters, the methanol inducible AOX1 promoter and the constitutive GAP promoter are most commonly used to drive gene expression. A set number of other promoters have been reported but not described in detail and were rarely applied by a broader public so far [4].
Bidirectional promoters provide divergent expression in opposing (forward and reverse) orientations. This enables coexpression of two genes by placing them in opposing orientations and placing a bidirectional promoter in between them (see FIG. 1 B, C).
There are no bidirectional promoters described in P. pastoris. However, bidirectional promoters have been studied in Saccharomyces cerevisiae and some information on natural bidirectional promoters and their function is available.
There are few examples for bidirectional promoters in S. cerevisiae that have been described in detail. Most prominently, the divergent organization of the GAL1-GAL10 promoter was studied. The GAL1 and GAL10 genes are organized in opposite orientations, with the intergenic region constituting a bidirectional promoter [5]. Both genes are required for the galactose metabolism and are tightly transcriptionally regulated by the carbon source. The genes on both sides are strongly induced on galactose and repressed on other carbon sources [6]. Therefore this bidirectional promoter provides similar expression levels on both sides and they share the same regulatory profile with a fixed ratio between the two sides. The bidirectional GAL1-GAL10 promoter has also been provided as an expression vector for bidirectional gene expression (pESC vector series, Stratagene/Agilent, La Jolla, Calif., USA). The GAL1-GAL10 promoter was also used to study a human heterodimeric transcription factor composed of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator [7]. In frame of this work also a constitutive bidirectional promoter was described by fusing the GPD and ADH1 promoters in opposite directions to each other. A similar fusion of constitutive promoters was performed by [8] using TEF1 and PGK1 in opposite orientations. The GAL1 and GAL10 sides of the GAL1-GAL10 promoter have furthermore been coupled with the constitutive GPD promoter, leading to bidirectional promoters with constitutive expression on one side and inducible expression on the other [9]. The two differently regulated sides did not influence each other and retained their regulatory profile and more than 85% of their monodirectional activity.
Another specific example of a bidirectional promoter in yeast is the UGA3-GLT1 intergenic region, which was shown to be affected by chromatin organization, but which was not tested as a promoter for bidirectional expression vectors [10].
Recent publications on genome wide analysis of natural bidirectional promoters in S. cerevisiae have shown that they are rather not involved in specific, high level expression, but rather in cryptic and pervasive transcription of the entire genome at low levels [11,12]. Namely, it was shown that weak pervasive transcription occurs in bidirectional fashion, and that the number of bidirectional promoters is significantly higher than previously estimated. Bidirectional pervasive transcription occurs not only next to protein coding sequences but also in intergenic regions [11]. These studies also suggested that bidirectionality is an intrinsic trait of eukaryotic promoters, leading in the majority of cases to short-lived unstable transcripts but also stable transcripts with a possible regulatory role [12]. The exact function of this pervasive bidirectional transcription is not fully understood, but they might play regulatory roles or help in maintaining chromatin structure [11].
Bidirectional promoters have also been studied in higher eukaryotes, namely in plants [13] and mammalian cells. Concerning mammalian cells bidirectional expression has been engineered using antibiotic regulated synthetic bidirectional promoters by tetracycline [14,15], pristinamycin [16] and two antibiotics at the same time (using a macrolide antibiotic on one side and a streptogramin antibiotic on the other side) [17]. Also sequence based approaches for promoter engineering of bidirectional promoters and natural bidirectional promoters were used in mammalian expression systems [18,19]. However, no library approach was applied so far to optimize expression by testing different bidirectional promoters to influence expression levels and ratios of coexpressed proteins.
Currently available bidirectional expression vectors rely on a bidirectional promoter flanked by two multiple cloning sites to clone in the genes to be expressed (FIG. 1 B). Although they facilitate cloning compared to dual gene expression with monodirectional promoters (FIG. 1 A), these vectors contain only a single bidirectional promoter. If different bidirectional promoters should be tested, a separate cloning vector is required for each promoter. Concomitantly this requires also multiple cloning steps for each gene pair into the vectors. Examples for such vectors have been mentioned above in S. cerevisiae [7,9] and there are bidirectional expression vectors available for mammalian cells (Clontech) and a specific, restriction site based screening vector for bidirectional elements [20]. However restriction sites with their palindromic sequences in front of the translation start can influence heterologous protein expression.
The Clontech vectors provide either bidirectional constitutive or bidirectional inducible expression with identical expression levels on both sides. These vectors have been optimized to facilitate the screening of a single gene. Therefore both sides provide identical expression. One side drives the expression of the GOI whereas the other side drives the expression of a reporter gene. If no activity assay or easy way of detection of the GOI is available, the reporter gene can help to screen for efficient expression of the GOI thereby avoiding the frequently applied fusion of the GOI to a fluorescent reporter protein.
Currently available bidirectional vectors [7,9] rely on a fixed bidirectional promoter and subsequent cloning steps using multiple cloning sites (MCS) (see FIG. 1 B and FIG. 2 A). Polson et al. [20] describe a vector that allows to test different promoters by restriction/ligation cloning which depends on the introduction of specific restriction sites at the end of each of the tested promoter sequences.
US20130157308A1 describes a bidirectional expression vector that can be utilized to determine the existence and characteristics of bidirectional promoters. The bidirectional expression vector includes two different reporter genes in a head to head (5′ to 5′) arrangement. In addition, the bidirectional expression vector can include a polylinker region located between the heads of the two reporter genes that provides multiple cloning sites for nonexclusive examination of polynucleotide sequences.
Currently used bidirectional promoters provide a very limited set of expression levels, ratios and regulatory profiles. In S. cerevisiae only five bidirectional promoters have been tested for expression vectors: 1) the natural GAL1-GAL10 promoter providing galactose inducible expression with the same strength on both sides, 2) a GPD and ADH1 fusion promoter [7], 3) a TEF1 and PGK1 fusion promoter [8] providing constitutive expression with the same strength on both sides and fusions of the 4) GAL1 sides with the GPD promoter and 5) the GAL10 side with the GPD promoter [9].
Notably these promoters provide only identical expression levels (strong expression) and a fixed ratio (approximately equal 1:1 ratio) on both sides [7] and the regulatory profiles are limited to constitutive expression and inducible expression using galactose.
Therefore, there are no bidirectional promoters that provide intermediate or low expression and with the currently known four promoters it is not possible to achieve different expression ratios of multiple genes to tune expression ratios for maximal yields of recombinant proteins or cellular metabolites from expressed pathways. Furthermore inducible expression can only be achieved using galactose. For example no auto regulatory bidirectional promoters and feedback loops are available for expression in yeasts.
Fine-tuning and optimizing the expression of a gene pair or multiple genes requires a broader scope of expression levels, ratios and time profiles.